Systematic delineation of optimal cytokine concentrations...

Systematic delineation of optimal cytokine concentrations to expand

hematopoietic stem/progenitor cells in co-culture with mesenchymal

stem cells

Pedro Z. Andrade,aFrancisco dos Santos,

aGraca Almeida-Porada,

b

Claudia Lobato da Silvaaand Joaquim M. S. Cabral

a

Received 28th October 2009, Accepted 12th March 2010

First published as an Advance Article on the web 27th April 2010

DOI: 10.1039/b922637k

The major obstacle to the widespread use of umbilical cord blood (UCB) in hematopoietic

stem/progenitor (HSC) cell therapy is the low cell dose available. A cytokine cocktail for the

ex vivo expansion of UCB HSC, in co-culture with a bone marrow (BM) mesenchymal stem cells

(MSC)-derived stromal layer was optimized using an experimental design approach. Proliferation

of total cells (TNC), stem/progenitor cells (CD34+) and colony-forming units (CFU) was assessed

after 7 days in culture, while sole and interactive effects of each cytokine on HSC expansion were

statistically determined using a two-level Face-Centered Cube Design. The optimal cytokine

cocktail obtained for HSC-MSC co-cultures was composed by SCF, Flt-3L and TPO

(60, 55 and 50 ng mL�1, respectively), resulting in 33-fold expansion in TNC, 17-fold in CD34+

cells, 3-fold in CD34+CD90+ cells and 21-fold in CFU-MIX. More importantly, these short-term

expanded cells preserved their telomere length and extensively generated cobblestone area-forming

cells (CAFCs) in vitro. The statistical tools used herein contributed for the rational delineation of

the cytokine concentration range, in a cost-effective way, while systematically addressing complex

cytokine-to-cytokine interactions, for the efficient HSC expansion towards the generation of

clinically significant cell numbers for transplantation.

Introduction

The efficient ex vivo expansion of hematopoietic stem/progenitor

cells (HSC), especially those from the umbilical cord blood

(UCB), which contains a limited numbers of bone marrow

repopulating cells per unit, would clearly widespread their

clinical applications to adult patients.1 The generation of

clinical significant cell numbers for a HSC transplant, while

maintaining their multilineage engraftment capability, would

potentially reduce the hematopoietic reconstitution time

and/or increase the engraftment success rate.2 The delineation

of protocols for expanding long-term engrafting CD34+ cells

would be of direct clinical utility, since most of the transplants

performed today with enriched hematopoietic progenitor cells

use CD34-enriched cells.3

In fact, a very recent study demonstrated that after a 10-day

ex vivo expansion, UCB CD34+ cells showed multilineage

repopulation after long-term and secondary engraftment in

sublethally irradiated NOD/SCID/IL-2R�/� mouse, comparable

to non-expanded HSC.4 Within the CD34+ cell fraction,

CD34+CD90+ cells, in particular, demonstrated to be efficient

in achieving rapid and sustained cell engraftment;5 indeed,

CD90 (Thy-1) expression has been recognized as superior to

CD38 (i.e. CD34+CD38�) and other markers as predictor of

the repopulating activity of CD34+ cells.6

Several ex vivo culture systems have been used with different

rates of success namely by testing different cytokine cocktails

in stroma-containing or stroma-free cultures with serum or in

serum-free conditions.7–11 Indeed, most cytokine combinations

tested include SCF, Flt-3L and TPO presumed to promote

extensive cell self-renewal and to limit levels of apoptosis.9,12

Nevertheless, numerous other molecules have been tested in

ex vivo HSC cultures: Zhang and co-workers obtained a

20-fold increase in SCID repopulating cells when IGF-binding

protein 2 and angiopoietin-like 5 were added to a cytokine

cocktail composed by SCF, FGF and TPO;13 Araki and

collaborators tested the chromatin modifying agents 5-aza-

20-deoxycytidine D (5-AzaD) and trichostatin (TSA) with

encouraging HSC expansion levels,14 whereas Peled and

co-workers reported significant enhancement of HSC proliferation

with lower levels of differentiation using the copper-chelator

agent, TEPA.15 In addition, in combination with other

cytokines, ex vivo expanded UCB CD133+ cells using

TEPA have been used in a phase I/II clinical trial, with high

engraftment rates.16

In our laboratory, we have previously established a serum-

free culture system using human BM MSC-derived feeder

layers, supplemented with SCF, Flt-3, bFGF and LIF, which

allowed an efficient expansion/maintenance of HSC from BM

and UCB.3,17 In particular for UCB, HSC-MSC cellular

interactions were shown to be crucial for the extensive expansion

of CD34+, CD34+CD38� cells and CFU-MIX in vitro.18

a IBB-Institute for Biotechnology and Bioengineering, Centre forBiological and Chemical Engineering, Instituto Superior Tecnico,Lisboa, Portugal. E-mail: [email protected];Fax: +351 218419062; Tel: +351 218419063

bDepartment of Animal Biotechnology, University of Nevada, Reno,NV, USA

This journal is �c The Royal Society of Chemistry 2010 Mol. BioSyst., 2010, 6, 1207–1215 | 1207

PAPER www.rsc.org/molecularbiosystems | Molecular BioSystems

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http://dx.doi.org/10.1039/B922637K

An effective ex vivo expansion protocol that would

maximize the cell output for a clinical application requires

multidimensional optimization, featuring proliferation/

differentiation, feeding regimen, product yield, quality and

operational costs.19,20 To this end, experimental designs have been

implemented, particularly regarding the optimization of cytokine

cocktails for the efficient generation of megakaryocytes,21

hematopoietic repopulating cells22 and UCB CD34+ cells23

in liquid cultures. Nevertheless, to our knowledge, no studies

focusing the systematic optimization of the cytokine cocktail

for such a complex HSC-MSC co-culture system are found in

the literature. Furthermore, particular cytokine interactive

effects have been reported in the literature, especially focusing

on SCF use with other hematopoietic cytokines,21,22,24

highlighting the importance of depicting epigenetic events

occurring in such a complex system.14

Here we used an experimental design approach to: (i) optimize

our previously established cytokine cocktail,3 which was

anticipated to exert its effect through a BM MSC-derived

stromal layer, for instance, by including TPO, while providing

a rational basis on the concentrations typically used; and (ii) to

determine the synergistic and sole effects of the cytokines used

towards the efficient generation of clinically relevant, quality-

controlled, cell numbers in a short-term culture period.

Materials and methods

Human donor cell preparation

The umbilical cord blood (UCB) samples were kindly provided

by Crioestaminal—Saude e Tecnologia, SA, and were

collected after maternal donor consent. Low density UCB

mononuclear cells (MNC) were separated by a Ficoll density

gradient (1.077 g ml�1) (GE Healthcare) and then washed

twice in Iscove’s Modified Dulbecco’s Medium (IMDM)

(GibcoBRL). UCB MNC were kept cryopreserved in liquid

nitrogen until further use.

Human bone marrow mesenchymal stem cell cultures

Bone marrow (BM) aspirates were harvested after informed

consent. BM MSC were isolated as previously described3,17

and kept in liquid nitrogen. Upon thawing, BM MSC were

expanded for 3–5 passages (3000 cells cm�2), using Dulbecco’s

Modified Essential Medium (DMEM) containing 10% Fetal

Bovine Serum (FBS) MSC-qualified (GibcoBRL), supplemented

with streptomycin (0.025 mg ml�1) and penicillin (0.025 U/ml)

(GibcoBRL), at 37 1C and 5% CO2 in a humidified

atmosphere. Medium was changed twice a week. Near cell

confluence (80–90%), cells were washed with phosphate saline

buffer (PBS, GibcoBRL) and detached from the flask by

adding Accutase (Sigma), for 7 min at 37 1C. In order to be

used as stromal feeder layers, cells were seeded in 24-well

plates (3000 cells cm�2) and grown until confluency. Before

performing co-cultures with UCB HSC, BM MSC were

treated with Mitomycin C (Sigma) (0.5 mg ml�1 solution

prepared in IMDM + 10% FBS) to prevent stromal over-

growth. The plates were incubated at 37 1C for 2.5 h. Next, cell

monolayers were washed twice with PBS for 5 min and fresh

DMEM 10% FBS was added. Co-cultures were established

within 24–48 h.

Ex vivo expansion of human CD34+-enriched cells

Upon thawing, UCB MNC were CD34+-enriched using

magnetic cell sorting (MACS, Miltenyi Biotec), and then

seeded in 24-well plates (3 � 104 cells mL�1) (on top of

confluent growth-inactivated BM MSC-derived stromal cell

layers) in QBSF-60 serum-free medium (Quality Biological,

Inc), at 37 1C and 5% CO2 humidified air, for 7–14 days

(cultures half-fed at day 7 and 10, depending on the

experiment).

The cytokines used to supplement culture medium were

SCF, Flt-3L, bFGF and TPO (Peprotech) and LIF (Chemicon),

tested in different combinations and/or concentrations. For

the validation studies (14 days cultures), two controls were

performed: (i) without exogenously added cytokines, with a

stromal layer (No Cyt) and (ii) without stromal layer, using the

most successful cytokine cocktail optimized by the experimental

design approach (No Stroma).

The cytokine-related costs of expanding TNC, CD34+ and

CD34+CD90+ cells (h/cells generated) in our co-culture

conditions were determined. For that we used the on-line

available pricelist for 2010 of the suppliers of human recombinant

SCF, Flt-3L, TPO and LIF. The % reduction was calculated

by dividing the amount saved by using Cocktail X (Y-X) per

the culture costs using cocktail Y.

Proliferative and phenotypic analysis

Both suspension and adherent fractions of hematopoietic cells

(detached by addition of Accutase, 6 min, 37 1C) were

determined by counting cell numbers using the Trypan Blue

(GibcoBRL) exclusion method for each different culture

condition. Fold increase (FI) in total nucleated cells (TNC)

was calculated by dividing the number of cells of each day by

the number of cells on day 0. Cells were also analyzed by flow

cytometry (FACSCalibur equipment, Becton Dickinson) using

a panel of monoclonal antibodies (FITC-, or PE-conjugated):

CD34 and CD90 for stem/progenitor cells; CD14, CD15 and

CD33 for myeloid lineage, CD7 (marker for early lymphopoiesis)

(Becton Dickinson Immunocytometry Systems) and CD41a

(for megakaryocytes). Isotype controls were also prepared for

every experiment. A minimum of 10000 events was collected

for each sample.

Clonogenic assays and cobblestone area-forming cells

Both fresh and expanded UCB CD34+ cells were characterized

in terms of clonogenic potential. The clonogenic assays were

performed in triplicate in MethoCult GF H4434 (Stem Cell

Technologies, Inc.). Cultures were maintained at 37 1C and

5% CO2 humidified air. After 14 days, colonies were counted

and evaluated accordingly to manufacturer’s instructions.

Colony-forming unit-granulocyte, macrophage (CFU-GM),

colony-forming unit-granulocyte, erythroid, macrophage,

megakaryocyte (CFU-Mix) and burst-forming unit-erythroid

(BFU-E) progenitors were identified. For the assessment of

cobblestone area-forming cells, expanded cells (2000 cells mL�1)

were cultured on top of a confluent (growth-inactivated)

1208 | Mol. BioSyst., 2010, 6, 1207–1215 This journal is �c The Royal Society of Chemistry 2010

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monolayer of MS-5 murine fibroblast cell line in long-term

culture medium (MyelocultTM, StemCell Technologies)

supplemented with 10�6 M hydrocortisone (Sigma) in 24-well

plates. Cultures were half-fed once a week and cobblestone

areas of more than 5 tightly packed cells underneath the

stromal layer were scored 2 weeks after seeding.25

Telomere length analysis

Relative telomere length (RTL) of fresh and expanded

CD34+-enriched cells was determined by telomere fluorescence

in situ hybridization and flow cytometry (flow FISH) using a

telomere-specific peptide nucleic acid (PNA) probe (Dako), as

described in ref. 26, using the 1301 cell line (Instituto Nazionale

per la Ricerca sul Cancro c/o CBA, Genova, Italy) as

control cells. Telomere length measurements were normalized

to day 0 values.

Experimental design

A two-level face-centered cube design (FC-CD) was performed

in order to optimize the concentrations of 4 factors: SCF,

Flt-3L, TPO and LIF, tested either at 0 ng mL�1 (low level, �1)or 100, 100, 50 and 10 ng mL�1 (high level, +1), respectively.

The concentration of bFGF was kept constant (5 ng mL�1),

since this growth factor was included in the original cocktail

basically to assure the maintenance of the stromal feeder layers

in the absence of serum.3 This FC-CD was composed by

27 runs: 16 factorial points, 3 replicated center points (that

provides an estimation of the experimental error27) and 8 axial

points. A second order model was obtained by fitting the

experimental data to eqn (1).

y = K + b1(x1) + b2(x2) + b3(x3) + b4(x4)

+ b1,2(x1,2) + b1,3(x1,3) + b1,4(x1,4) + b2,3(x2,3)

+ b2,4(x2,4) + b3,4(x3,4) + b1,1(x1)2 + b2,2(x2)

2

+ b3,3(x3)2 + b4,4(x4)

2

(1)

where y is the response measured (fold increase in TNC,

CD34+ cells, CFU-MIX, CFU-GM at day 7), bi the regressioncoefficients corresponding to the main effects, bi,j the

coefficients for the second order interactions and bi,i the

quadratic coefficients.

Statistical analysis

The determination of the regression coefficients followed a

sequential backward elimination procedure, where the least

significant terms (p > 0.05) of the eqn (1), in each step, were

eliminated and absorbed into the error. Nevertheless, in the

case of significant second order interactions or quadratic

coefficients, the eliminated terms corresponding to the main

effects were reintroduced in the model (as described in

ref. 21, but only if Lack of Fit test remained non-significant

(p > 0.05)).

Validation results are presented as mean� standard error of

mean (SEM). Comparisons between experimental results were

determined by Mann-Whitney test for independent samples,

when appropriate. A p-value less than 0.05 was considered

statistically significant.

Results

We have previously established a BM MSC-derived stromal-

based system for the successful expansion of hematopoietic

stem/progenitor cell ex vivo using a serum-free culture medium

supplemented with SCF, Flt-3L, bFGF and LIF.3 Here our

goal is to rationally delineate the optimal cytokine concentrations

for the successful expansion of UCB HSC, in a stromal-based

system. In particular, we aim to further improve the rate of

expansion of UCB HSC by altering the cytokine cocktail

previously studied, not only in terms of the concentrations

used (ng mL�1), but also by adding TPO to the initial cocktail.

Indeed, in order to maintain the stem cell pool and prevent

apoptosis in vitro, cytokine cocktails typically composed by

SCF, Flt-3, TPO among others, such as LIF and/or IL-6 have

been reported.3,9,12,17,22

FC-CD for the optimization of hematopoietic stem/progenitor

cell expansion

UCB CD34+ enriched (94.1 � 0.8%) hematopoietic

stem/progenitor cells (initial cell density: 3 � 104 cells mL�1)

were cultured on top of confluent BM MSC-derived stromal

feeder layers (2.8 � 0.6 � 103 cells/well), for 7 days, using the

cytokine cocktails presented in Table 1, according to a two-

level Face Centered Cube Design.27 Based on our previous

knowledge,3,17,18 as well as cocktails reported in the literature,

we established the range of the study between 0 and 10 ng ml�1

of LIF/50 ng mL�1 of TPO/100 ng mL�1 of SCF and Flt-3L.

This strategy allowed the statistical determination of the

main effects of SCF, Flt-3L, LIF and TPO on hematopoietic

stem/progenitor cell expansion, as well as all second-order

interactions, present in eqn (1), for each of the 4 responses

measured: fold increase in TNC, CD34+ cells, CFU-GM and

CFU-MIX. The regression coefficients, their p-value and the

summary of fitting (R2 and Lack of Fit test) for each of these

responses are presented in Table 2.

The second order polynomials generated for each

response described a significant percentage of the experimental

data (0.87 > R2 > 0.74), with no Lack Of Fit associated

(0.38 > p > 0.12) (Table 2).

Table 2 presents the statistically significant parameter values

of eqn (1), as well as the summary of the fit for each model.

The statistical analysis of our data showed that LIF does not

have a significant impact in any of the responses (both main

and interactive effects)—p > 0.05. In addition, it is possible to

observe that Flt-3L main effect is not significant for any of the

responses (p > 0.163), while SCF was non-significant only for

the expansion of CFU-MIX (p = 0.291). Nevertheless, for

both cases, since the quadratic terms (Flt-3L2 and SCF2)

were significant (p o 0.01), the first order coefficients were

reintroduced into the model. Interestingly, for all of the

responses measured, the second order terms SCF2 and

Flt-3L2 were negative indicating a downward concavity of

the models, suggesting the existence of a maximum value in the

cytokine concentration range in study (Fig. 1).

As a result of the models established, the optimal cytokine

concentrations for each response were graphically determined

and are presented in Table 3. The predicted SCF concentrations

that maximize the responses range from 52 to 68 ng mL�1,


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while Flt-3L varies from 52 to 55 ng mL�1. Since TPO had a

positive main effect (in the absence of a significant second

order term), the maximum concentration tested (50 ng mL�1)

also maximizes the expansion of hematopoietic stem/progenitor

cells, although the optimum value for this cytokine should be

located outside of the range tested (>50 ng mL�1). Interestingly,

all the optimized cocktails are roughly similar, which clearly

indicates that the responses measured are highly correlated.

Validation

The determined optimized cocktail for the expansion of CD34+

cells (Z9) was then validated against three other conditions, as

presented in Table 4: the previous cytokine cocktail used in

our lab (CR5)3 and two controls—in the absence of cytokines

(No Cyt) and without stroma (No Stroma).

The HSC-MSC co-cultures were performed in a 14-day

period, being analyzed at days 3, 7, 10 and 14 in terms of

TNC, the hematopoietic stem/progenitor phenotypes CD34+

and CD34+CD90+, and for the more committed progenitors

from the early lymphoid (CD7+), myeloid (CD14+, CD15+

and CD33+) and megakaryocytic lineages (CD41a+ cells)

(Fig. 2 and Table 5). In addition, the clonogenic potential of

both fresh and expanded cells was assessed by CFU-MIX and

CFU-GM assays (Fig. 3a and b), as well as their ability to

form cobblestone areas (CAFC) and cell relative telomere

length (RTL) (Fig. 4a and b).

In agreement with the model predictions, Z9 cocktail

provided the highest fold increases in TNC (26�2-fold) andCD34+ cells (16 � 1-fold) after 7 days in culture (Fig. 2a and b)

(p o 0.05). Most importantly, Z9 combination led to the

highest expansion of the most primitive CD34+CD90+ cells

(3.8 � 0.7-fold) and CAFC (26.4 � 4.3) (p o 0.05), while

retaining their relative telomere length (97 � 3%) (Fig. 4a and

b) after one-week culture period. When compared with our

previous cytokine cocktail (CR5),3 Z9 provided significantly

higher stem/progenitor cell expansion levels (p o 0.05),

representing an optimization factor of 1.7 for TNC and

CD34+ cell expansion, and a 2.7 factor for CD34+CD90+

cell expansion. In addition, the lower levels of cytokines

required in Z9 along with higher HSC productivities, led to

Table 1 Experimental design—Face centered composite design:A—Design matrix for the optimization of the cytokine cocktail:SCF, Flt-3L, LIF and TPO. B—Cube representation of thedesign used in the present studies. SCF, Flt-3L, TPO and LIFwere tested either at 0 ng mL�1 (low level, �1) or 100, 100, 50and 10 ng mL�1 (high level, +1), respectively. Mid level (0) =50 ng mL�1 for SCF and Flt-3L, 25 ng mL�1 for TPO and5 ng mL�1 for LIF

SCF Flt-3L LIF TPO

+ + + ++ + + —+ + — ++ + — —+ — + ++ — + —+ — — ++ — — —— + + +— + + —— + — +— + — —— — + +— — + —— — — +— — — —0 0 0 00 0 0 00 0 0 00 0 + 00 0 — 00 0 0 +0 0 0 —0 + 0 00 — 0 0+ 0 0 0— 0 0 0

Table 2 Parameter values of eqn (1) and summary of fit for the fold increase in TNC, CD34+ cells, CFU-GM and CFU-MIX. Only statisticallysignificant terms (p o 0.05) were considered for the model. Nevertheless, non-significant main effects were reintroduced into the model when thesecond order terms were significant. All models did not present Lack of Fit (p > 0.05)

Term

FI (TNC) FI (CD34+ cells) FI (CFU-GM) FI (CFU-MIX)

Value P value Value P value Value P value Value P value

K �1.037 o0.01 �0.045 o0.01 �0.395 o0.01 �0.846 o0.01bSCF 0.613 o0.01 0.394 o0.01 0.219 o0.01 0.317 0.291bFlt-3L 0.405 0.163 0.223 0.200 0.146 0.165 0.260 0.465bTPO 0.097 0.039 0.069 0.046 0.056 0.031 0.004 0.095bSCFxSCF �0.005 o0.01 �0.003 o0.01 �0.002 o0.01 �0.003 o0.01bFlt-3LxFlt-3L �0.004 o0.01 �0.002 o0.01 �0.001 o0.01 �0.002 o0.01R2 0.87 0.82 0.74 0.87RMSEa 4.7 3.5 2.9 2.7Lack of fit test p-value 0.12 0.38 0.31 0.19

a RMSE—Root mean square error.


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a reduction of 47, 55 and 62% in cytokine-related culture costs

for the generation of TNC, CD34+ and CD34+CD90+ cells,

respectively, in a 7-day period.

As expected, hematopoietic cells cultured either in the

absence of the stromal feeder layer (No Stroma) or without

cytokines (No Cyt) did not present any significant expansion of

TNC (when compared to any of the other conditions—po 0.05),

being roughly similar at day 14 (16-fold and 12-fold,

respectively). Interestingly, theNo Cyt condition presented higher

CAFCs expansion levels (although non-significant; p > 0.05),

when compared to the No Stroma condition, especially at day 14

(2.7 � 1.4 and 0.2 � 0.1, respectively) (Fig. 4a).

The differentiative potential of the expanded cells was

studied during time in culture, testing for the early lymphocytic

Fig. 1 3-D representation of the Face-centered composite design (FCCD) as a function of Flt-3L and SCF. (a): Fold increase in TNC. (b): Fold

increase in CD34+ cells. (c): Fold increase in CFU-MIX. (d): Fold increase in CFU-GM. TPO was set at 50 ng mL�1.

Table 3 Optimized cytokine concentrations for the expansion of TNC, CD34+ cells, CFU-MIX and CFU-GM and respective maximumexpansion reached. The cytokine cocktails that maximized each response were determined graphically and code-named. For TNC and CD34+ cellexpansion, Z9—60 ng mL�1 of SCF, 55 ng mL�1 of Flt-3L and 50 ng mL�1 of TPO; for the CFU-MIX, M1—52 ng mL�1 of SCF and Flt-3L and50 ng mL�1 of TPO; for CFU-GM, G1—68 ng mL�1 of SCF, 54 ng mL�1 of Flt-3L and 50 ng mL�1 of TPO

Response Code name SCF/ng mL�1 Flt-3L/ng mL�1 TPO/ng mL�1 Optimized FI

CD34+ cells Z9 60 55 50 21TNC Z9 60 55 50 33CFU-MIX M1 52 52 50 17CFU-GM G1 68 54 50 13

Table 4 Validation of the model predictions. Four conditions were tested: the optimized cocktail for the expansion of TNC and CD34+ cells (Z9),the previously used cocktail (CR5) and two controls—in the absence of cytokines (No Cyt) and using Z9 cocktail in the absence of stroma(No Stroma)

Code name SCF/ng mL�1 Flt-3L/ng mL�1 TPO/ng mL�1 LIF/ng mL�1

Z9 60 55 50 —CR5 100 100 0 0.1No Cyt — — — —No Stroma (Z9) 60 55 50 —


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(CD7+ cells), myeloid (CD14+, CD15+ and CD33+ cells)

and megakaryocytic (CD41a+ cells) lineages (Table 5). Z9 and

CR5 conditions were able to successfully maintain/expand

these progenitors in agreement with our previous results.3,17

As expected by the absence of TPO, CR5 cocktail resulted in

significantly lower levels of the megakaryocytic progenitor

cells CD41a+ (0.8 � 0.1% and 1.3 � 0.1% at days 7 and 14)

when compared to Z9 (2.3� 0.8 and 1.3� 0.1% at days 7 and 14,

respectively) (p o 0.05).

In terms of clonogenic potential, Z9 presented higher expan-

sion rates of CFU-MIX (106� 10-fold at day 14) and CFU-GM

(488 � 9-fold) when compared to CR5 (49 � 4 and 389 � 17,

respectively; po 0.05), especially later in culture, consistent with

the observed expansion of TNC and CD34+ cells (Fig. 2a and b),

as well as with the differentiative potential of the expanded cells

shifted mainly towards the myeloid lineage, as assessed by the

immunophenotypic analysis (Table 5).

Discussion

We have previously reported that the expansion/maintenance

of HSC from both BM and UCB is highly favored in the

presence of BM MSC-derived feeder layer from human origin

in a cytokine supplemented (SCF + Flt-3L + LIF + bFGF)

serum-free culture system.3,17 Particularly for UCB HSC, we

showed that the ex vivo expansion is not only dependent on the

presence of the feeder layer but also on the cellular interactions

established in the co-culture, using a cytokine cocktail

which was anticipated to exert their effect through stromal

or accessory cells.3,28

In the present study, our goal was to optimize our

previously established cytokine cocktail, while providing a

rational basis on the concentrations to be used, by using an

experimental design. We focused on the generation of clinically

significant cell numbers in a short-term (1 week) period, as the

optimal time for an efficient static liquid culture should be

limited to 7–14 days.19 To our best knowledge, no systematic

optimization on the cytokine cocktail composition was

performed in a HSC-MSC co-culture system. In particular,

we tested if we could further improve the rate of expansion of

UCB stem/progenitor cells in our stromal-based culture system

by including to the early acting SCF and Flt-3L, TPO, a

cytokine described as efficient at promoting the viability of the

more primitive cells, while suppressing apoptosis.12,29

A FC-CD was performed in which 4 factors (SCF, Flt-3L,

TPO and LIF) in two different levels were correlated with the fold

expansion of both TNC, CD34+ cells, CFU-MIX and CFU-GM

after 7 days in co-culture with BM MSC. bFGF concentration

was not included as a target of our optimization strategy, since

this factor was included basically for the maintenance of the

stromal feeder layers in the absence of serum.3

Consistent with other reports, SCF, Flt-3L and TPO

exhibited statistically significant positive effects on the expansion

of HSC.12 Interestingly, for all the responses measured, the

second order terms SCF2 and Flt-3L2 were found to be

negative and highly significant, leading to the determination of

a local maximum in the range of our study (0–100 ng mL�1),

due to the downward concavity of the resulting models.

Biologically, these negative second order terms probably mean

a significant inhibition of hematopoietic stem/progenitor cell

growth when large amounts of SCF and/or Flt-3 are added to

the cultures, suggesting a substrate inhibition-like effect, typically

found in biological reactions. In addition, our design allowed

the estimation of interactive cytokine effects, which have been

reported in the literature, especially involving SCF with

other cytokines.14,21,22,30 Although none of the interactions

determined were statistically significant, for any of the

responses measured, there is an improvement in the response

by using increasing concentrations of SCF and Flt-3L, resulting in

an optimized cytokine cocktail composed by SCF (60 ng mL�1),

Flt-3L (55 ng mL�1) and TPO (50 ng mL�1)—Z9, for the

Fig. 2 Validation of the model predictions. Fold increase in (a) TNC

(n = 6), (b) CD34+ (n = 6) and (c) CD34+CD90+ (n = 6) cells after

two weeks in HSC-MSC co-culture supplemented with the cytokine

cocktails present in Table 4. No phenotypic analyses were performed

for theNo Stroma condition due to the low amounts of cells generated.

Results are presented as Mean � SEM. * p o 0.05.


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expansion of TNC, and CD34+ cells, after 7 days in co-culture.

These model-predicted values were validated in another set of

experiments and represent a 2-fold higher cell productivity

with significant reduction in culture costs (50–65%) due to the

substantial reduction in cytokine concentrations, when com-

pared with our previously used cytokine cocktail.3 In addition,

Z9 combination provided a 4-fold expansion of the more

primitive CD34+CD90+ cells14 after 7 days in culture, while

retaining their telomere length and the ability to form cobble-

stone areas in vitro. Telomere length and telomerase activity

constitute strong evidence of the quality of the expanded

graft.31 Consistent with our results, Gammaitoni and co-workers

reported telomere length maintenance and up-regulation of

telomerase activity in long-term cultures of UCB, BM and

mobilized peripheral blood (MPB) CD34+ cells, with a

20-fold increase in CAFCs after 4 weeks in co-culture with

OP9 cells.32 Interestingly, in our studies, hematopoietic cells

cultured in the presence of stroma, non cytokine-supplemented,

provided consistently higher expansion levels of CAFCs, when

compared with cells grown without feeder layer, supplemented

with our optimized cytokine cocktail (Z9). These results

suggest a strong effect of the BM-derived stroma on the

maintenance/expansion of CAFCs,33 described as a measure

of SCID repopulating ability.34 We reason that BM MSC-

based feeder cell layer, even in the absence of exogenously

added cytokines, is able to produce growth factors such as

SCF, LIF and Flt-3L.35 The produced growth factors,

although at a basal level, together with the cell-to-cell

interactions provided by the feeder cells,28 are able to preserve

a subset of the more primitive long-term repopulating cells,

here assessed by the CAFC content, during the two-week

culture period.36

Fig. 3 Clonogenic Potential of expanded cells supplemented with the cytokine cocktails presented in Table 4: the optimal cytokine cocktail

obtained in this study (Z9), our previous cytokine cocktail (CR5) and two controls, without cytokines (No Cyt) and in the absence of stroma,

using Z9 cocktail (No Stroma). Fold increase in CFU-MIX (a) and CFU-GM (b), during time in culture for each condition, are presented as

values � SEM (n = 4; *p o 0.05).

Fig. 4 Expansion of umbilical cord blood hematopoietic cells in co-culture with BM MSC using different cytokine combinations, present in

Table 4. The fold increase in cobblestone area-forming cells (CAFCs) (a) and the relative telomere length (b) were determined during time in

culture for Z9, CR5, No Cyt and No Stroma conditions. Results are presented as Mean � SEM (n = 4; *p o 0.05).

Table 5 Differentiative potential of expanded cells with the cytokine cocktails Z9 and CR5. Results are presented as percentages � SEM (n= 4).* po 0.05. Non-adherent cells were harvested periodically and analyzed by flow cytometry with CD7 FITC, CD14 PE, CD15 FITC, CD33 PE andCD41a PE antibodies. Isotype control antibodies were used to determine the level of non-specific binding. Due to the limiting cell number, nodifferentiative potential was attained both for the No Stroma and No Cyt conditions

Time (days)

% CD7+ % CD14+ % CD15+ % CD33+ % CD41a+

Z9 CR5 Z9 CR5 Z9 CR5 Z9 CR5 Z9 CR5

0 8.5 � 0.9 7.3 � 2.5 11.5 � 1.8 88.4 � 3.7 1.4 � 0.210 40.0 � 4.0 38.2 � 6.4 24.1 � 4.3 16.7 � 2.4 46.2 � 3.9 46.7 � 7.9 87.7 � 3.6 92.6 � 1.3 2.3 � 0.8 (*) 0.8 � 0.1 (*)14 46.6 � 3.1 48.9 � 3.0 23.5 � 4.3 25.1 � 2.1 52.5 � 4.6 49.4 � 4.8 93.8 � 1.4 89.2 � 1.9 5.8 � 0.7 (*) 1.3 � 0.1 (*)


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In the present FC-CD studies (7 days), LIF presented a

positive main effect on HSC-MSC co-cultures, though without

statistical significance. Nevertheless, since previous results by

our group suggest that LIF might have a more pronounced

effect on progenitor expansion, especially later in culture,18,28

ongoing studies are currently being performed in our lab to

depict the precise mechanism of action of LIF in the present

co-culture system.

The differentiative potential of the expanded cells using the

different cocktails was primarily shifted towards the myeloid

lineage, as assessed by immunophenotypic analysis and

clonogenic potential studies, as previously reported, while

maintaining a significant percentage of cells with an early

lymphocytic potential (CD7+ cells).3,37 As expected by the

absence of TPO, the CR5 cocktail3 yielded the lowest levels of

the megakaryocytic progenitor cells CD41a+ when compared

to Z9 (with TPO). Indeed, it has been widely reported

in the literature the classical strong effect of TPO on

thrombopoiesis.38

In summary, we have successfully optimized a cytokine

cocktail for the ex vivo expansion of UCB HSC in co-culture

with BM MSC, in a one-week time period, using an experi-

mental design approach. In addition, our results contribute for

the rational delineation of the concentration range of the

cytokines to be used in a HSC expansion protocol, in a

systematic and cost-effective way, while quantitatively addressing

the complex interactions among cytokines/growth factors.

More importantly, the expanded cells maintained their telomere

length and extensively originated cobblestones in vitro. This

work offers important clues to better understand the cellular

determinants underlying ex vivo expansion of HSC, providing

the basis for the establishment of efficient and controlled

culture systems for the generation of clinically significant cell

numbers in the settings of BM transplantation using UCB

expanded cells.

Acknowledgements

The authors would like to thank to Crioestaminal—Saude e

Tecnologia, SA for UCB samples.

This work was financially supported by MIT-Portugal

Program and grants SFRH/BD/38720/2007 and SFRH/BD/

38719/2007 awarded to Pedro Z. Andrade and Francisco dos

Santos from Fundacao para a Ciencia e a Tecnologia,

Portugal.

The authors declare no conflict of interests.

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Systematic delineation of optimal cytokine concentrations...

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